Si Hyeock Lee

Genome sequences of the human body louse and its primary endosymbiont provide insights into the permanent parasitic lifestyle

As an obligatory parasite of humans, the body louse (Pediculus humanus humanus) is an important vector for human diseases, including epidemic typhus, relapsing fever, and trench fever. Here, we present genome sequences of the body louse and its primary bacterial endosymbiont Candidatus Riesia pediculicola. The body louse has the smallest known insect genome, spanning 108 Mb. Despite its status as an obligate parasite, it retains a remarkably complete basal insect repertoire of 10,773 protein-coding genes and 57 microRNAs. Representing hemimetabolous insects, the genome of the body louse thus provides a reference for studies of holometabolous insects. Compared with other insect genomes, the body louse genome contains significantly fewer genes associated with environmental sensing and response, including odorant and gustatory receptors and detoxifying enzymes. The unique architecture of the 18 minicircular mitochondrial chromosomes of the body louse may be linked to the loss of the gene encoding the mitochondrial single-stranded DNA binding protein. The genome of the obligatory louse endosymbiont Candidatus Riesia pediculicola encodes less than 600 genes on a short, linear chromosome and a circular plasmid. The plasmid harbors a unique arrangement of genes required for the synthesis of pantothenate, an essential vitamin deficient in the louse diet. The human body louse, its primary endosymbiont, and the bacterial pathogens that it vectors all possess genomes reduced in size compared with their free-living close relatives. Thus, the body louse genome project offers unique information and tools to use in advancing understanding of coevolution among vectors, symbionts, and pathogens.

Biochemical and Molecular Analysis of Deltamethrin Resistance in the Common Bed Bug (Hemiptera: Cimicidae)

Abstract This study establishes deltamethrin resistance in a common bed bug, Cimex lectularius L., population collected from New York City (NY-BB). The NY-BB population was 264-fold more resistant to 1% deltamethrin in contact bioassay compared with an insecticide-susceptible population collected in Florida (FL-BB). General esterase, glutathione S-transferase, and 7-ethoxycoumarin O-deethylase activities of NY-BB were not statistically different from those of FL-BB. cDNA fragments that encoded the open reading frame of voltage-sensitive sodium channel α-subunit genes from the FL-BB and NY-BB populations, respectively, were obtained by homology probing polymerase chain reaction (PCR) and sequenced. Sequence alignment of the internal and 5′ and 3′ rapid amplification of cDNA ends (RACE) fragments generated a 6,500-bp cDNA sequence contig, which was composed of a 6,084-bp open reading frame (ORF) encoding 2,027 amino acid residues and 186-bp 5′ and 230-bp 3′ untranslated regions (5′ and 3′ UTRs, respectively). Sequence comparisons of the open reading frames of the α-subunit genes identified two point mutations (V419L and L925I) that were presented only in the NY-BB population. L925I, located the intracellular loop between IIS4 and IIS5, has been previously found in a highly pyrethroid-resistant populations of whitefly (Bemisia tabaci). V419L, located in the IS6 transmembrane segment, is a novel mutation. A Val to Met mutation at the corresponding position of the bed bug V419, however, has been identified in the tobacco budworm as a kdr-type mutation. This evidence suggests that the two mutations are likely the major resistance-causing mutations in the deltamethrin-resistant NY-BB through a knockdown-type nerve insensitivity mechanism.

A point mutation in a glutamate‐gated chloride channel confers abamectin resistance in the two‐spotted spider mite, Tetranychus urticae Koch

The molecular mechanisms and genetics of abamectin resistance mediated by target site insensitivity in the two‐spotted spider mite, Tetranychus urticae, were investigated by comparing two isogenic abamectin‐susceptible (AbaS) and abamectin‐resistant (AbaR) strains. Cloning and sequencing of full‐length cDNA fragments of γ‐amino butyric acid (GABA)‐gated chloride channel genes revealed no polymorphisms between the two strains. However, sequence comparison of the full‐length cDNA fragment of a T. urticae glutamate‐gated chloride channel gene (TuGluCl) identified a G323D point mutation as being tentatively related with abamectin resistance. In individual F2 progenies obtained by backcrossing, the G323D genotype was confirmed to correlate with abamectin resistance. Bioassays using progeny from reciprocal crossings revealed that the abamectin resistance trait resulting from TuGluCl insensitivity is incompletely recessive.

Molecular, biochemical and histochemical characterization of two acetylcholinesterase cDNAs from the German cockroach Blattella germanica.

Full length cDNAs encoding two acetylcholinesterases (AChEs; Bgace1 and Bgace2) were cloned and characterized from the German cockroach, Blattella germanica. Sequence analyses showed that both genes possess all the typical features of ace, and that Bgace1 is orthologous to the insect ace1 whereas Bgace2 is to the insect ace2. Transcript level of Bgace1 was significantly higher (c. 10 fold) than that of Bgace2 in all 11 tissues examined, suggesting that Bgace1 likely encodes a predominant AChE. Multiple AChE bands were identified by native polyacrylamide gel electrophoresis and isoelectricfocusing from various tissue preparations, among which ganglia produced distinct two major and two minor AChE bands, indicative of the presence of at least two active AChEs. B. germanica AChEs appeared to be mainly localized in the central nervous system as demonstrated by histochemical activity staining, together with quantitative analysis of Bgace transcripts. Fluorescence in situ hybridization of the 1st thoracic ganglion confirmed that Bgace1 is predominantly transcribed and further showed that its transcript is found in almost entire region of inter or motor neurones including the cell bodies and axonal/dendritic branches. Bgace2 transcript is found only in the subset of neurones, particularly in the cell body. In addition, certain neurones were observed to express Bgace1 only.

Decreased detoxification genes and genome size make the human body louse an efficient model to study xenobiotic metabolism.

The human body louse, Pediculus humanus humanus, has one of the smallest insect genomes, containing ∼10 775 annotated genes. Annotation of detoxification [cytochrome P450 monooxygenase (P450), glutathione‐S‐transferase (GST), esterase (Est) and ATP‐binding cassette transporter (ABC transporter)] genes revealed that they are dramatically reduced in P. h. humanus compared to other insects except for Apis mellifera. There are 37 P450, 13 GST and 17 Est genes present in P. h. humanus, approximately half the number found in Drosophila melanogaster and Anopheles gambiae. The number of putatively functional ABC transporter genes in P. h. humanus and Ap. mellifera are the same (36) but both have fewer than An. gambiae (44) or Dr. melanogaster (65). The reduction of detoxification genes in P. h. humanus may be a result of this louses simple life history, in which it does not encounter a wide variety of xenobiotics. Neuronal component genes are highly conserved across different insect species as expected because of their critical function. Although reduced in number, P. h. humanus still retains at least a minimum repertoire of genes known to confer metabolic or toxicokinetic resistance to xenobiotics (eg Cyp3 clade P450s, Delta GSTs, B clade Ests and B/C subfamily ABC transporters), suggestive of its high potential for resistance development.

Brief exposures of human body lice to sublethal amounts of ivermectin over-transcribes detoxification genes involved in tolerance

Transcriptional profiling results, using our non‐invasive induction assay {short exposure intervals (2–5 h) to sublethal amounts of insecticides [

Sodium channel mutations associated with knockdown resistance in the human head louse, Pediculus capitis (De Geer)

Pyrethroid resistance in human head louse populations is widespread in the United States and worldwide. We previously documented that the knockdown resistance of permethrin-resistant head louse populations is associated with the T929I and L932F (T917I and L920F in the numbering of the louse amino acid sequence) mutations in the voltage-sensitive sodium channel α-subunit gene. In order to identify additional sodium channel mutations potentially associated with knockdown resistance, we cloned and sequenced full-length cDNA fragments from insecticide-susceptible (Ecuador) and permethrin-resistant (Florida) head louse populations and from an insecticide-susceptible body louse population (Israel). Sequence comparisons of the complete open reading frames of the sodium channel genes identified one additional novel mutation (M815I), which was located in the IIS1-2 extracellular loop of the α-subunit, from the permethrin-resistant head louse population. Absolute conservation of the Met815 residue at the corresponding positions within sodium channels from all known susceptible populations of insect species implied that the M815I mutation likely has a functional significance in resistance. Sequence analyses of cloned cDNA fragments and genomic DNA fragments from individual louse samples, both containing the three mutation sites, confirmed that all the mutations exist en bloc as a haplotype. Northern blot analysis identified a single 7.2 kb transcript. The comparison of complete open reading frame sequences (6156 bp) of sodium channel gene between head and body lice revealed 26 polymorphic nucleotides, of which only one resulted in a conservative amino acid substitution (glutamic versus aspartic acid at 11th amino acid position). The virtual identity in nucleotide sequences indicated that both body and head lice are conspecific, and lends justification of the use of the body louse as a surrogate organism for the head louse in biochemical and molecular biology studies. Conserved point mutations resulting in knockdown resistance to the pyrethrins, the pyrethroids, and DDT are suitable for detection by various DNA-diagnostic protocols for monitoring and resistance management.

Which acetylcholinesterase functions as the main catalytic enzyme in the Class Insecta

Most insects possess two different acetylcholinesterases (AChEs) (i.e., AChE1 and AChE2; encoded by ace1 and ace2 genes, respectively). Between the two AChEs, AChE1 has been proposed as a major catalytic enzyme based on its higher expression level and frequently observed point mutations associated with insecticide resistance. To investigate the evolutionary distribution of AChE1 and AChE2, we determined which AChE had a central catalytic function in several insect species across 18 orders. The main catalytic activity in heads was determined by native polyacrylamide gel electrophoresis in conjunction with Western blotting using AChE1- and AChE2-specific antibodies. Of the 100 insect species examined, 67 species showed higher AChE1 activity; thus, AChE1 was considered as the main catalytic enzyme. In the remaining 33 species, ranging from Palaeoptera to Hymenoptera, however, AChE2 was predominantly expressed as the main catalytic enzyme. These findings challenge the common notion that AChE1 is the only main catalytic enzyme in insects with the exception of Cyclorrhapha, and further demonstrate that the specialization of AChE2 as the main enzyme or the replacement of AChE1 function with AChE2 were rather common events, having multiple independent origins during insect evolution. It was hypothesized that the generation of multiple AChE2 isoforms by alternative splicing allowed the loss of ace1 during the process of functional replacement of AChE1 with AChE2 in Cyclorrhapha. However, the presence of AChE2 as the main catalytic enzyme in higher social Hymenoptera provides a case for the functional replacement of AChE1 with AChE2 without the loss of ace1. The current study will provide valuable insights into the evolution of AChE: which AChE has been specialized as the main catalytic enzyme and to become the main target for insecticides in different insect species.

Extensive gene duplication of acetylcholinesterase associated with organophosphate resistance in the two-spotted spider mite.

Monocrotophos‐resistant two‐spotted spider mites (TSSMs), Tetranychus urticae, are known to possess three mutations on the acetylcholinesterase (AChE) gene (Tuace) that are involved in target site insensitivity. Cross‐strain comparison of three strains (highly resistant AD, moderately resistant PyriF and susceptible UD strains) revealed that resistant strains have relatively more Tuace copies than the UD strain and that the levels of transcript were directly proportional to copy numbers. AChEs from the AD and PyriF strains had similar Vmax values to those of AChE from the UD strain but increased Km and reduced kcat constants, suggesting that the mutated, resistant form of AChE may carry a fitness cost. Relative copy numbers of Tuace in field populations varied from 2.4 to 6.1, correlating well with their levels of resistance (r2= 0.895). These results are suggestive of the involvement of Tuace gene duplication in resistance. Thus, monocrotophos resistance in TSSMs appears to have evolved through a combination of mutation accumulation and extensive gene duplication.

Three mutations identified in the voltage-sensitive sodium channel α-subunit gene of permethrin-resistant human head lice reduce the permethrin sensitivity of house fly Vssc1 sodium channels expressed in Xenopus oocytes

Point mutations in the para-orthologous sodium channel alpha-subunit of the head louse (M815I, T917I, and L920F) are associated with permethrin resistance and DDT resistance. These mutations were inserted in all combinations using site-directed mutagenesis at the corresponding amino acid sequence positions (M827I, T929I, and L932F) of the house fly para-orthologous voltage-sensitive sodium channel alpha-subunit (Vssc1(WT)) gene and heterologously co-expressed with the sodium channel auxiliary subunit of house fly (Vsscbeta) in Xenopus oocytes. The double mutant possessing M827I and T929I (Vssc1(MITI)/Vsscbeta) caused a approximately 4.0mV hyperpolarizing shift and the triple mutant, Vssc1(MITILF)/Vsscbeta, caused a approximately 3.2mV depolarizing shift in the voltage dependence of activation curves. Vssc1(MITI)/Vsscbeta, Vssc1(TILF)/Vsscbeta, and Vssc1(MITILF)/Vsscbeta caused depolarizing shifts ( approximately 6.6, approximately 7.6, and approximately 8.8mV, respectively) in the voltage dependence of steady-state inactivation curves. The M827I and L932F mutations reduced permethrin sensitivity when expressed alone but the T929I mutation, either alone or in combination, virtually abolished permethrin sensitivity. Thus, the T929I mutation is the principal cause of permethrin resistance in head lice. Comparison of the expression rates of channels containing single, double and triple mutations with that of Vssc1(WT)/Vsscbeta channels indicates that the M827I mutation may play a role in rescuing the decreased expression of channels containing T929I.